Microbiology II. Methanogenesis. Christopher Bräsen. Methanococcus jannaschii. Methanosarcina barkeri

Transkript

1 Microbiology II Methanogenesis Methanococcus jannaschii Christopher Bräsen Methanosarcina barkeri

2 Lecture Plan Mikrobielle Physiologie I Energetik Bräsen Mikrobielle Physiologie II Einige Prinzipien und Mechanismen im zentralen Kohlenstoffmetabolismus Bräsen Keine Vorlesung Bräsen Mikrobielle Physiologie III Nitrat Atmung Bräsen Mikrobielle Physiologie IV Acetogenese und der Acetyl CoA/Kohlenmonoxid Dehydrogenase Weg Bräsen Mikrobielle Physiologie V Anaerobe Nahrungskette und Methanogenese Bräsen Mikrobielle Physiologie VI Sulfate Reduktion Bräsen Antibiotika (Penicillium notatum) Meckenstock Mikroorganismen in der Umwelt (Geobacter metallireducens) Meckenstock Mikrobielles Wachstum (Elusimicrobium minutum) Meckenstock Mikrobielle Fortbewegung (Thioploca) Meckenstock Viren (T4) Meckenstock Geschichte der Mikrobiologie Meckenstock Wrap up/ausweichtermin Meckenstock/Bräsen

3 Questions 4 What is Acetogenesis, what are the substrates, what the products? Name the underlying pathway for CO 2 reduction and the two branches involved? What is the key coenzyme? What is the key enzyme and which reaction does it catalyze? How is energy gained? Which condition is crucial for autotrophic Acetogenesis? Outline the steps of the anaerobic food chain! (remember also the different modes of primary fermentations, substrates, products etc.) What are the substrates and products of secundary fermentations? What is the key problem and how are these reactions driven? What is Syntrophy?

4 Fragen 4 Was ist Acetogenese, was sind die Substrate, was die Produkte? Benenne den zugrunde liegenden Stoffwechselweg zur CO 2 Reduktion und dessen beiden Zweige, die daran beteiligt sind? Wie heißt das Schlüsselcoenzym dieses Weges? Wie heißt das Schüsselenzym und welche Reaktion katalysiert es? Wie hoch ist die Energieausbeute? Welche Bedingung ist für die autotrophe Acetogenese essentiell wichtig? Skizzieren Sie die Schritte der anaeroben Nahrungskette! (remember also the different modes of primary fermentations, substrates, products etc.) Was sind die Substrate und Produkte sekundärer Fermentationen? Was ist bei diesen Reaktionen das Grundproblem und wie werden sie angetrieben? Was ist Syntrophie?

5 Anaerobic food chain Methane is a major end product of anaerobic biomass degradation only in anoxic environments where the concentrations of sulphate, nitrate, Mn(IV) or Fe(III) are low. In the presence of these electron acceptors, methanogenesis is out competed by anaerobic respiration, mainly for thermodynamic reasons. (Thauer RK et al. (2008) Nature Reviews Microbiology 6, ) Brock Mikrobiologie Pearson 2013

6 Methanogenesis CO 2 + 4H 2 ~ 1.5 ATP CH H 2 O ΔG 0 = 131 kj/mol (ph 2 1 atm) Methanococcus jannaschii Acetate + H + CH 4 + CO 2 0,5 1 ATP ΔG 0 = 36 kj/mol Strictly anaerobic Euryarchaea Methanobacteriales, Methanopyrales, Methanococcales, Methanomicrobiales, Methanosarcinales Most methanogens can grow with CO 2, formate and H 2 Methanosarcinales also with acetate (methylamine, methanol) Unusual coenzymes 70% of the methane is formed from acetate Methanosarcina barkeri Brock Mikrobiologie Pearson 2013

7 Methanogenesis CO 2 + 4H 2 ~ 1.5 ATP CH H 2 O ΔG 0 = 131 kj/mol (ph 2 1 atm) Methanococcus jannaschii Acetate + H + CH 4 + CO 2 0,5 1 ATP ΔG 0 = 36 kj/mol Methane is a very effective green house gas! Methanococcus jannaschii Methanosarcina barkeri Brock Mikrobiologie Pearson 2013

8 Methanogenesis from CO 2 and H 2 CO 2 + 4H 2 CH H 2 O ~ 1.5 ATP (1/2 ATP) ΔG 0 = 131 kj/mol (ph 2 1 atm) ΔG = 131 kj/mol kj/mol x lg ([P1] a [P2] b )/([S1] c [10 5 ] 4 ) = 131 kj/mol kj/mol x 20 = ~ 17 kj/mol (ph atm) ~1/3 ATP Carbonate respiration In natural habitats ph 2 is as low as 10 5 atm due to methanogenic activity This is the lower limit for (autotrophic) methanogenesis CO 2 fixation via the rcodh/acs pathway (=reductive acetyl CoA pathway) Allows however for syntrophic H 2 production

9 Methanogenesis from CO 2 and H 2 chemolithoautotrophic CO 2 + 4H 2 CH H 2 O ~ 1.5 ATP (1/2 ATP) ΔG 0 = 131 kj/mol (ph 2 1 atm) ΔG = ~ 17 kj/mol (ph atm) ~1/3 ATP Oxidative part Reductive part 4H 2 CO 2 8 [H] ~ 1.5 ATP (1/2 ATP) Via Electron transport phosphorylation (ETP) 2 H 2 O CH 4

10 Methanogenesis Electron transfer + Energy generation Electron transfer C1 transfer Methyl group reduction Quinone derivative Electron transport Strictly anaerobic Euryarchaea Methanobacteriales Methanopyrales, Methanococcales, Methanomicrobiales, Methanosarcinales Most methanogens can grow with CO 2, formate and H 2 Methanosarcinales also with acetate (methylamine, methanol) Unusual coenzymes F 420 absorbs light at 420 nm and fluoresce blue green

11 Methanogenesis from CO 2 and H 2 CO 2 + 4H 2 CH H 2 O ~ 1.5 ATP (1/2 ATP) ΔG 0 = 131 kj/mol, (ph 2 1 atm) ΔG = ~ 17 kj/mol (ph atm) Methanosarcinales with cytochromes and Methanophenazin! higher growth yield higher H 2 threshold MFR = Methanofuran H 4 MPT = Tetrahydromenthanopterin CoM SH = Coenzym M CoB SH = Coenzym B Heterodisulfid Thauer et al. (2008) Nature Reviews Microbiology 6,

12 Tetrahydrofolate Tetrahydromethanopterin

13 Methanogenesis from CO 2 and H 2 Formyl methanofuran DH ΔµH + 2 H + Fd red H 2 + Fd ox CO 2 + 4H 2 CH H 2 O ~ 1.5 ATP (1/2 ATP) Formyl tetrahydromethanopterin cyclohydrolase ΔG 0 = 131 kj/mol, (ph 2 1 atm) ΔG = ~ 17 kj/mol (ph atm) ΔµNa + 2 Na + (4 H + ) H 2 H 2 Methenyltetrahydromethanopterin reductase Methylenetetrahydromethanopterin reductase Methyltransferase (B 12 cofactor) Methyl CoM reductase ΔµH + 4 H + H 2 Hydrogenase/ Heterodisulfide reductase complex

14 Methanogenesis from CO 2 and H 2 CO 2 + 4H 2 CH H 2 O ~ ATP (1/3 ATP) ΔG 0 = 131 kj/mol, (ph 2 1 atm) ΔG = ~ 17 kj/mol (ph atm) Methanobacteriales, Methanococcales, Methanopyrales and Methanomicrobiales without cytochromes and Methanophenazin! Lower growth yield Lower H 2 threshold Thauer et al. (2008) Nature Reviews Microbiology 6,

15 Methanogenesis from acetate Acetate + H + CH 4 + CO 2 Acetate ~ ATP 1 2 ATP 1 2 ADP + P i O ΔG 0 = 36 kj/mol Disproportionation: Oxidation of the carboxyl group to CO 2 Reduction of the methyl group to CH 4 Transport of 7 H + (Na + )/acetate corresponding to ~2 ATP Oxidative part CoA S~C CH 3 Reductive part But: Activation of acetate to acetyl CoA requires at least 1 ATP Carbonic anhydrase: CODH/ACS 2 [H] ~ 1/2 1 ATP Via Electron transport phosphorylation (ETP) CO 2 CH 4 O CoA S~C CH 3

16 Methanogenesis from acetate Acetate + H + CH 4 + CO 2 ~ ATP CODH/ACS Disproportionation: ΔG 0 = 36 kj/mol Oxidation of the carboxyl group to CO 2 Reduction of the methyl ΔµNa group to CH + 4 Transport of 6 8 H + (Na + )/acetate ΔµNa + corresponding to 1,5 2 ATP But: Activation of acetate ΔµH + to acetyl CoA requires at least 1 ATP Carbonic anhydrase: Ferredoxin: Methanophenazin oxidoreductase Methyltransferase (B 12 cofactor) Methyl CoM reductase Hydrogenase/ Heterodisulfide reductase complex

17 Methanogenesis from acetate Acetate + H + CH 4 + CO 2 ~ ATP CODH/ACS ΔG 0 = 36 kj/mol Disproportionation: Oxidation of the carboxyl group to CO 2 Reduction of the methyl 4 Na + Ferredoxin: Methanophenazin oxidoreductase group to CH 4 Transport of 6 8 H + (Na + )/acetate 2 Na + corresponding to 1,5 2 ATP But: Activation of acetate to acetyl CoA requires at least 1 ATP Carbonic anhydrase: 2 H + Methyltransferase (B 12 cofactor) Methyl CoM reductase Hydrogenase/ Heterodisulfide reductase complex

18 Brock Mikrobiologie Pearson 2013 Anaerobic food chain

19 Sulfate reduction Competition of sulfate reducers with syntrophic methanogenic communities and acetogens for fermentation products (lactate, propionate, acetate, H 2 in anaerobic environments) Sulfate reducers with broader substrate spectrum than e.g. methanogens Higher affinity and lower threshold for H 2 sulfate reducers outcompete methanogens at elevated SO 4 2 concentrations e.g. in marine sediments Muyzer G & Stams AJM, 2008

20 Life style Energiequelle Licht Redoxreaktion Photo Chemo Elektronendon(at)or Litho Organo anorganischer Stoff organischer Stoff trophie Kohlenstoffquelle anorganischer Stoff Autoorganischer Stoff Hetero Microorganisms show a high metabolic diversity Play important roles in the biogeochemical cyles of elements

21 CO 2 Fixation

22 Reductive acetyl CoA pathway reductive CODH/ACS pathway = Wood Ljungdal pathway ~ 1 ATP + 2 NAD(P)H (F 420 H 2 ) + 3 ferredoxin/ Σ pyruvate Acetyl CoA synthase/co dehydrogenase Strict anaerobes: Methanogenic and sulfate reducing Euryarchaea, acetogens, some proteobacteria a.o., sulfate reducers Acetyl CoA, pyruvate Well suited for the assimilation of C1 units Pyruvat CO 2 + Fd red Fd ox Anabolism

23 Reductive pentose phosphate cycle (Calvin Benson cycle) RubisCO = Ribulose bisphosphate carboxylase/oxygenase 7ATP +5 NAD(P)H/ Σ pyruvate From light dependent reactions of photosynthesis (ETP reactions in chemosynthesis) 2 Pyruvat 4 ATP 2 NAD(P)H Plants, algae, cyanobacteria, most aerobic and facultativ aerobic (chemolithoautotrophic) Bacteria, anoxygen phototrophic purple bacteria Triosephosphates, 3 phosphoglycerate, sugar phosphates as intermediates

24 The Citric acid cycle (oxidative) in aerobic organisms Acetate activation (two ATP equivalents) Two irreversible steps < 30 kj/mol 320 mv 190 mv Energy dissipation + 30 mv 500mV 320 mv +113 mv APS/SO mv, SO 3 2 /HS 110 mv! Thauer RK (1988) Citric acid cycle, 50 years on. Eur J Biochem 176:

25 Reductive citric acid cycle (Arnon Buchanan cycle) 2 ( 3) ATP + 3 NAD(P)H + 2 ferredoxin/ Σ pyruvate ATP and ferredoxin from light dependent reactions of photosynthesis (ETP reactions in chemosynthesis) Fumarate reductase 2 Oxoglutarate synthase ATP citrate lyase Pyruvate synthase Isocitrate dehydrogenase Anaerobic Green sulfur bacteria (Chlorobiales) and other Proteobacteria, Aquificales (microaerophilic) Acetyl CoA, pyruvate, oxaloacetate, succinyl CoA, 2 oxoglutarate, (PEP) Advantages under anaerobic, microaerophilic conditions

26 Hydroxypropionate bi cycle ~ 7 ATP + 6 NAD(P)H ( 1QH 2 ) / Σpyruvate

27 3 Hydroxypropionate bi cycle ~ 7 ATP + 6 NAD(P)H ( 1QH 2 ) / Σ pyruvate Acetyl CoA carboxylase 2x Malyl CoA lyase Propionyl CoA Anaerobic Chloroflexaceae carboxylase Acetyl CoA, pyruvate, succinyl CoA Suited for mixotrophic assimilation of fermentation products under oligotrophic conditions

28 Dicarboxylate/4 Hydroxybutyrate cycle 3 Hydroxypropionate/4 Hydroxybutyrate cycle Dicarboxylat/ 4 Hydroxybutyrate cycle Anaerobic Crenarchaeota (Thermoproteales, Desulfurococcales 3 Hydroxypropionate/ 4 Hydroxybutyrate cycle Aerobic Crenarchaeota (e.g. Sulfolobus) and Thaumarchaeota (Thermoproteales, Desulfurococcales ~ 5 ATP + 2 Fd red + NAD(P)H + 4 [H] / Σ pyruvate ~ 6 ATP + 3 NAD(P)H + 4 [H] / Σ pyruvate

29 Questions 5 What is methanogenesis? What are the substrates, what the products? Which organisms perform methanogenesis? Name a special feature of methanogens/methanogenesis. How does methanogenesis from acetate basically works? What is oxidized, what is reduced? What is especially challenging with respect to energetics? How does chemolithotrophic methanogens fix CO 2? Which are the energy gaining reactions in both types of methanogenesis? What is the lower ph 2 limit of methanogenesis and what does that mean for syntrophy? Name at least 3 pathways of CO 2 fixation!

30 Fragen 5 Was ist Methanogenese? Was sind die Substrate, was die Produkte? Welche Organismen betreiben Methanogenese? Benennen Sie spezielle Eigenschaften der Methanogenen. Wie verläuft grundsätzlich die Methanogenese aus Acetat? Was wird oxidiert, was reduziert? Was ist im Hinblick auf die Energetik eine besondere Herausforderung? Wie fixieren chemolithoautotrophe Methanogene CO 2? Welches sind die energieliefernden Reaktionen der in beiden Formen der Methanogenese? Was ist das untere Limit des H 2 Partialdruckes für die Methanogenese und was bedeutet das für die Snytrophie Benennen Sie mindestens 3 Wege zur CO 2 Fixierung

31 Übung: Mikrobielles Wachstum I Aufgabe: 1.) E. coli wurde auf eine OD 600 von 0,1 aus einer exponentiell wachsenden Kultur auf das gleiche Medium (Minimalmedium mit 20 mm als C & E Quelle) überimpft und wächst exponentiell mit einer Verdopplungszeit von 40 min. Wie lange dauert es, bis die Kultur eine OD 600 von 5,0 erreicht hat? 2.) Ihre Kurve soll in etwa alle 0.5 OD Einheiten einen Meßpunkt zeigen; zu welchen Zeitpunkten müssen Sie Probe nehmen?

32 Übung: Mikrobielles Wachstum I Aufgabe: 1. E. coli wurde auf eine OD 600 von 0,1 aus einer exponentiell wachsenden Kultur auf das gleiche Medium (Minimalmedium mit 20 mm als C & E Quelle) überimpft und wächst exponentiell mit einer Verdopplungszeit von 40 min. Wie lange dauert es, bis die Kultur eine OD 600 von 5,0 erreicht hat? Wachstumsrate µ: Verdoppelungszeit t D : μ ln 0 0 ln 2 μ

33 Übung: Mikrobielles Wachstum I Wachstumsrate µ: Verdoppelungszeit t D : μ ln 0 0 ln 2 μ 0 ln ln 0 μ μ, 0, ,,,, 1, 1,, 1 0,, min

34 Übung: Mikrobielles Wachstum I Wachstumsrate µ: Verdoppelungszeit t D : μ ln 0 0 ln 2 μ 0 ln ln 0 μ 0 μ.,,,, 1, 1,, 1 95 min, 0, ,, 1,, 1,, 1,, 1,, 1 40 min,, 1 23 min 17,2 min 13,1 min 6,2 min (OD 4.5 OD 5)

35 Übung: Mikrobielles Wachstum I

36 Übung: Mikrobielles Wachstum II Wie entwickelt man ein Medium?

37 Übung: Mikrobielles Wachstum II Minimalmedium (definierte Zusammensetzung), C limitiertes Wachstum (heterotroph) 1 g TZ/ 1 g C ~50% der C & E Quelle gehen in den Baustoffwechsel Wie viel Stickstoff (NH 4 Cl in mm) muss dem Medium mindestens zugegeben werden, damit der Stickstoff bei einer gegebenen C & E Quelle (20 mm Glucose) nicht limitierend wird?